Efficacy of probiotics/synbiotics supplementation in patients with chronic kidney disease: a systematic review and meta-analysis of randomized controlled trials

Background Chronic kidney disease (CKD) is a serious and steadily growing health problem worldwide. Probiotic and synbiotic supplementation are expected to improve kidney function in CKD patients by altering imbalanced intestinal flora, regulating microbiota metabolites, modulating the brain-gut axis, and reducing inflammation. Objectives Our aim is to report the latest and largest pooled analyses and evidence updates to explore whether probiotic and synbiotic have beneficial effects on renal function and general conditions in patients with CKD. Methods We conducted a systematic literature search using PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials from inception until 1 December 2023. Eligible literatures were screened according to inclusion and exclusion criteria, data were extracted, and a systematic review and meta-analysis was performed. Measurements included renal function-related markers, inflammatory markers, uremic toxins, lipid metabolism-related markers and electrolytes levels. Results Twenty-one studies were included. The results showed that probiotic/synbiotic significantly reduced blood urea nitrogen (BUN) (standardized mean difference (SMD), −0.23, 95% confidence interval (CI) −0.41, −0.04; p = 0.02, I2 = 10%) and lowered c-reactive protein level (CRP) (SMD: −0.34; 95% CI: −0.62, −0.07; p = 0.01, I2 = 37%) in CKD patients, compared with the control group. Conclusion In summary, probiotic/synbiotic supplementation seems to be effective in improving renal function indices and inflammation indices in CKD patients. Subgroup analyses suggested that longer-term supplementation is more favorable for CKD patients, but there is a high degree of heterogeneity in the results of partial subgroup analyses. The efficacy of probiotic/synbiotic in treating CKD needs to be supported by more evidence from large-scale clinical studies. Systematic review registration https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42024526836, Unique identifier: CRD42024526836.


Introduction
CKD is characterized by abnormalities in kidney structure or function that persist for a period of at least 3 months (1).The clinical features of CKD include decreased renal function and/or increased urinary albumin excretion (proteinuria) (2).It is a disease that progresses slowly over time, and a significant number of patients eventually reach end-stage renal disease and require dialysis for treatment (3,4).Currently, the global prevalence of CKD is about 11%, and with increasing age, the prevalence in people over 70 years old is as high as 34% (5,6).In addition, patients with CKD have an increased risk of cardiovascular disease, hypertension, diabetes, and infections (7).Although CKD has received considerable attention from scientists and clinicians, the care and treatment of the condition are still not up to par (8).Consequently, there is a pressing need to explore new drugs or therapeutic approaches.
Previous studies have shown that ecosystem imbalances are strongly associated with a number of chronic diseases, such as chronic kidney disease, diabetes and cardiovascular disease (9,10).There is also evidence showed that imbalances in the gut microbiota may contribute to CKD (11).In addition, worsening CKD can further exacerbate imbalances in gut flora (12).Researchers proposed the gut-kidney axis and the CKD-colon axis in 2011 (13) and 2015 (14) to characterize the interactions between the kidney and the gut.Probiotics, synbiotic supplements are then expected to slow the progression of CKD by regulating the balance of intestinal flora (15).Probiotics, consisting of active microorganisms, colonize the human intestinal tract to improve the microbiological balance and benefit human health (16).Recent studies indicated that probiotics could potentially offer advantages to individuals with chronic kidney disease (17)(18)(19).The current definition of synbiotic has been updated to "a mixture of live microorganisms and substrates selectively utilized by host microorganisms to provide health benefits to the host" (20).There have been a number of studies aimed at evaluating the role of synbiotic supplementation in patients with CKD (21)(22)(23)(24).Currently, non-food probiotics and synbiotic supplements are becoming increasingly available in the United States (25).
The use of probiotics has shown potential as a nutritional strategy for the prevention and/or treatment of CKD.In some animal studies, Lactobacillus supplementation has been shown to slow the progression of chronic kidney disease and delay renal failure by altering shortchain fatty acid and nicotinamide metabolism (26).In addition, an exploratory clinical study found that serum levels of tumor necrosis factor-α (TNF-α), Interleukin (IL)-6, IL-18, and endotoxin were significantly reduced in patients with CKD after probiotics administration (27).Despite growing interest in the potential role of probiotics in improving chronic kidney disease, there is a lack of extensive cross-sectional studies to comprehensively assess the effect of probiotics/synbiotics on the general condition of CKD patients in the population.In addition, although there have been previous metaanalyses of the relationship between probiotics and CKD, the outcome indicators of these analyses have focused on one of many metrics, such as kidney function or metabolism (28,29).Therefore, a systematic review and meta-analysis incorporated latest RCT studies was designed to comprehensively investigate the effects of probiotics/ synbiotics supplementation on renal function, lipid metabolism, inflammation, uremic toxin levels and electrolyte levels in dialysis/ non-dialysis CKD patients.

Search strategy
The review program was established by two investigators (LC) and (WW) prior to the start of the study and registered with the PROSPERO International Prospective Registry of Systematic Reviews (registration number CRD42024526836).This study was conducted according to the Cochrane Manual and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (30).Two independent reviewers (LC and YLT) searched PubMed, Embase, Web of Science and Cochrane Library from inception until December 2023.We searched the databases using the following terms: "probiotics, " "probiotic, " "synbiotics, " "synbiotic, " "renal insufficiency, chronic, " "chronic renal insufficiencies, " "renal insufficiencies, chronic, " "chronic renal insufficiency, " "kidney insufficiency, chronic, " "chronic kidney insufficiencies, " "kidney insufficiencies, chronic, " "chronic kidney diseases, " "chronic kidney disease" "disease, chronic kidney, " "diseases, chronic kidney, " "kidney disease, chronic, " "kidney diseases, chronic, " "chronic renal diseases, " "chronic renal disease, " "disease, chronic renal, " "diseases, chronic renal, " "renal disease, chronic, " "renal diseases, chronic" and "chronic kidney insufficiency." Two researchers independently searched and evaluated the included studies, and any disagreements in the literature search were resolved by conferring with a third researcher (WW).Specific search strategies are shown in Supplementary Table 1.

Eligible criteria
The study met all of the following criteria (1) study design: randomized controlled study; (2) study participants: patients with a confirmed diagnosis of chronic kidney disease; (3) intervention: the intervention group should receive any dose of probiotic or synbiotic supplementation; (4) comparison regimen/control group: participants in the control group may receive a placebo or other medication and if other medications are used in the treatment group, they also control group must be used in the same way; (6) language: articles published in English.
Studies were excluded for the following reasons: (1) they were reviews, meta-analyses, case reports, conference abstracts, and guidelines; (2) the study was animal-based; (3) the study was published in a language other than English.

Research screening
After excluding duplicate records, two researchers independently screened the titles and abstracts of all identified records to remove irrelevant documents.A full-text review was then conducted to determine eligibility for inclusion.Any disagreements regarding study selection could be resolved through discussion with a third researcher (LC, YLT, and WW).The study selection process is shown in Figure 1.

Data extraction
The following data were extracted from the included studies: (a) The basic information, including first author, publication year, region, When continuous variables in the study were reported as median with range or interquartile range, we calculated the mean ± standard deviation through the validated mathematical method.When data were missing or not reported in the study, we contacted the corresponding authors to obtain completed data if available.

Quality assessment
Quality assessment of eligible RCTs was performed according to the Cochrane Handbook for Systematic Reviews of Interventions 5.1.0,based on seven terms: randomized sequence generation, allocation concealment, participant and personnel blinding, blinding of outcome assessment, incomplete outcome data, selective reporting and other biases sources (31).Three outcomes were assessed for each study, including low risk, high risk, and unclear risk.Studies with more "low risk" of bias assessment were considered superior.

Statistical analysis
Evidence synthesis was performed in Review Manager 5.4 version (Cochrane Collaboration, Oxford, United Kingdom).The SMD was applied for the comparison of continuous variables.All metrics were reported with mean ± SD.The heterogeneity in studies was assessed through the inconsistency index (I 2 ).I 2 > 50% were considered as significant heterogeneity.A random-effect model was used to estimate the combined SMD when significant heterogeneity was detected (I 2 > 50%).Otherwise, the fixed-effect model was applied.In addition, we performed one-way sensitivity analyses to evaluate the effect of included studies on the combined results for outcomes with significant heterogeneity.Subgroup analyses were used to explore sources of heterogeneity.The subgroup analysis has not been conducted for indicators which were few included in literatures.Because the limited number of literatures may lead to a significant discrepancy between subgroups, which could impact the accuracy of the results.Publication bias was evaluated visually by creating funnel plots via Review Manager 5.4 version (Cochrane Collaboration, Oxford, United Kingdom), as well as by conducting Egger's regression tests using Stata 15.0 version (Stata Corp, College Station, TX, United States) for outcomes with 5 or more included studies.p-value < 0.05 was considered as statistically significant publication bias.

Literature search
The initial search was completed on 1 December 2023.We have identified 310 potentially relevant publications from PubMed, 434 from Embase, 103 from The Cochrane library, 540 from Web of science and 2 from manual retrieval.Endnote was used to eliminate duplicate publications, resulting in 791 records for review.After excluding publications that did not meet the inclusion criteria, we included 21 studies for systematic review and meta-analysis.A flow diagram illustrating the exclusion of articles with specific reasons is shown in Figure 1 (PRISMA flowchart).

Study characteristics
We conducted a systematic review and meta-analysis of 869 patients with chronic kidney disease involved in 21 RCT studies (17-19, 21, 22, 32-47).The sample size ranged between 11 and 80, and the mean age of the patients was recorded ranged from a minimum of 45 years old to a maximum of 76 years old.The majority of the patients were from Asia.The articles were studied from 2013 to 2023 (Table 1).

Risk of bias assessment
The risk of bias assessment is presented in Supplementary Figures S1, S2.Most of the included studies were considered to have a low or unclear risk of bias.Two papers had significant baseline imbalance (17,36).The main source of bias in two pieces of literatures was the failure to implement double blinding (21,44).Six literatures were at high risk because of poor completeness of data results (19, 32-34, 44, 46).
Subgroup analysis based on patient population revealed no changes in indoxyl sulfate level after evaluation with either

Change in blood calcium
Six publications (19,21,34,37,40,46) involving 227 patients (114 probiotics/synbiotics patients; 113 control patients) were included in the analysis regarding blood calcium levels, and the results suggested that there were no statistically significant differences (SMD: 0.21; 95% CI: −0.05, 0.47; p = 0.11) and no significant heterogeneity (I 2 = 0%, p = 0.84) in the change of blood calcium levels in patients in the probiotics/synbiotics group compared with the control group (Figure 7A).It is worth noting that funnel plots (Figure 6E) and Egger's test revealed significant publication bias (p = 0.049).

Change in blood potassium
Two hundred and sixteen patients (110 probiotics/synbiotics patients; 106 control patients) originating from seven publications Frontiers in Nutrition 14 frontiersin.org(21, 32-34, 40, 41, 46) were included in the analysis regarding blood potassium levels.And the results suggested that there were no statistically significant differences (SMD: −0.1; 95% CI: −0.38, 0.18; p = 0.48) and no significant heterogeneity (I 2 = 8%, p = 0.36) in the change of blood potassium levels of the patients in the probiotics/ synbiotics group compared with the control group (Figure 7B).Funnel plot (Figure 6F) and Egger's test did not reveal significant publication bias (p = 0.426).Frontiers in Nutrition frontiersin.org

Sensitivity analysis
Because the comprehensive analysis of HDL showed significant heterogeneity, we conducted one-way sensitivity analyses for comparison of HDL to evaluate the influence of each individual study on the combined SMD through removing the individual study one by one.Sensitivity analyses revealed that the new combined SMD remained constant after exclusion of any individual study for HDL (Figure 8).

Findings from meta-analysis
The gut microbiota consists of more than 100 trillion bacteria and plays an important role in normal body functions, particularly in immune and metabolic homeostasis.There is growing evidence that alterations in the gut microbiota can affect multiple organ systems and lead to many chronic diseases such as CKD (48).CKD is a serious and steadily growing health problem worldwide.As a progressive disease, the majority of CKD patients are referred to dialysis treatment, and effective pharmacological treatments are still being explored (49).One of the promising drug candidates is the modification of dysbiotic gut flora through probiotics/synbiotics supplementation to reduce levels of gut-derived uremic toxins and reduce chronic microinflammation thereby improving renal function (50).Due to the small number of published studies, it is still highly controversial whether this probiotics/synbiotics intervention affects renal function, uremic toxicity, and inflammation levels in patients with CKD.In this study, we systematically compiled and analyzed the clinical evidence of RCT on probiotics/synbiotics for the treatment of CKD to provide better guidance for clinical practice.
Our results showed that probiotic/synbiotics supplementation of CKD patients can decreased BUN in CKD patients, and no significant heterogeneity was found in the analysis results, which to a certain extent reflects the effect of probiotic/synbiotics to improve renal function in CKD patients.However, the use of probiotic/synbiotics had no effect on Sensitivity analysis of HDL.We also stratified patients according to region, duration of treatment, age, and whether or not they were receiving dialysis treatment.Subgroups were analyzed for several important indicators: urea nitrogen, serum creatinine, CRP, indoxyl sulfate, and blood phosphorus.The results of subgroup analyses suggested that probiotics/ synbiotics were more effective in reducing BUN in non-hemodialysis CKD patients.This probably due to the fact that CKD in hemodialysis patients usually has progressed to the stage of end-stage renal disease, and the renal units are irreversibly damaged, which is difficult to be improved by drug treatment (51).The results of subgroup analysis also showed that probiotics/synbiotics had a better effect on CRP reduction in non-hemodialysis CKD patients, suggesting that probiotics/synbiotics had a better effect on the improvement of inflammation in non-hemodialysis patients.In CKD patients, increased inflammation is associated with several negative clinical outcomes such as increased oxidative stress, vascular dysfunction and increased risk of cardiovascular disease, as the ameliorative effect of probiotics/synbiotics on inflammation in non-hemodialysis patients is beneficial to patients (52,53).And one of the goals of treatment for hemodialysis patients is to reduce inflammation, thus effectively improving the survival of these patients (54).Meanwhile, the results of the subgroup analysis stratified on the basis of the duration of probiotics/synbiotics treatment showed that there is a significant reduction in BUN and CRP levels in CKD patients when probiotics/synbiotics are applied for a longer period of time compared to the control group.This suggested that adherence to probiotics/synbiotics for a longer period of time is more favorable for CKD patients.Interestingly, when subgroup analyses were performed on a regional basis, we found that probiotics/synbiotics supplementation was more effective in reducing BUN in Asian patients and in reducing CRP in patients from the Europe.The number of studies that included patients from the Europe was small, so the related results need to be further verified.

Possible mechanisms
The relationship between probiotics/synbiotics and CKD has been recognized with the increasing understanding of the health effects of microbial balance on the host.Essentially, probiotics/synbiotics supplementation can modulate the imbalance of the gut microbiota for the biosynthesis of targeted compounds with bioactive properties in CKD patients (55).At the same time, probiotics improves the integrity of the intestinal epithelial barrier and reduces the production of uremic toxins to some extent (56,57).With the fluctuation of gut bacteria, probiotics can modulate inflammation by establishing a balance between pro-inflammatory and anti-inflammatory cytokines in the body (58).In addition, metabolites from the gut microbiota also play an important role in maintaining homeostasis in the gut for the benefit of host health through fermentation of amino acids and dietary fiber, production of vitamins and neurotransmitters, and modification of bile acids (59).For example, Zhu et al. (60) showed that short-chain fatty acids (SCFAs) from a variety of bacteria reduced the expression of genes for inflammatory cytokines, chemokines, and pro-fibrotic proteins in diabetic kidneys, which, in turn, reduced proteinuria, glomerular hypertrophy, pedunculated cell injury, and interstitial fibrosis in mice with acute kidney injury and CKD.Indeed, probiotic or synbiotics supplementation may also reverse the expansion of harmful gut microbes that produce excess uremic toxins and attenuate the development of CKD (32, 61).

Strengths and limitations 4.3.1 Limitations
Firstly, most of the publications included in this meta-analysis were RCT cohort studies.The sample sizes of RCT studies are small, and potential bias from small samples is unavoidable.Secondly, the main population groups of the studies we included were from Asia, with fewer people from other states, and there may be regional selectivity bias.Whether the results can be generalized to other regions needs to be confirmed by further studies.Thirdly, the heterogeneity of the studies included in the analysis of HDL was large, which may hinder the robustness of the results.In addition, the RCTs involved in the study did not report adverse events in patients, so adverse events were not included in the study.

Strengths
Firstly, this study is the latest meta-analysis of probiotics/ synbiotics for CKD with the largest sample size available.Secondly, the original studies included in this article were all RCTs, which were of high quality, with good study design and a balanced baseline.Thirdly, this study confirmed that probiotics/synbiotics had an ameliorative effect on renal function and inflammatory status in patients with CKD, which has been consistent with previous studies.Fourthly, compared with previous meta-analyses, our study included a wider range of outcome indicators and incorporated the most recent RCT studies, thus allowing for the most up-to-date evidence on probiotics/synbiotics supplementation in CKD treatment.What's more, this study provides more options and guidance notes for clinical CKD treatment.

Conclusion
In conclusion, this is the latest systematic review and metaanalysis demonstrating that probiotic/synbiotics interventions reduced BUN and CRP in patients with CKD, although there was insufficient evidence of a positive effect of probiotics/synbiotics on lipids and blood electrolytes.Regarding BUN and CRP, the results of our meta-analysis emphasize the positive effects of probiotic/ synbiotics supplementation using longer (≥3 months) treatment durations in Asian patients.This area deserves further research to elucidate the mechanism of probiotics/synbiotics for the possible treatment of CKD and to further assess the safety of different types of probiotics/synbiotics through randomized controlled trials.

TABLE 1
Baseline characteristics of include studies and methodological assessment.
and serum creatinine, indicators renal function.And the analysis results of eGFR and serum creatinine did not show apparent heterogeneity.This result may be due to the fact that eGFR and serum creatinine is strongly influenced by ethnicity, gender and age.And the baseline could not be standardized across studies.In addition to this, the use of probiotics/synbiotics can reduce CRP expression levels and improve inflammatory status in CKD patients.However, probiotic/ synbiotic supplementation did not significantly alter blood electrolyte levels and lipid metabolism-related markers in CKD patients compared with placebo.No significant publication bias was detected by Egger's test and funnel plot for all indicators except blood calcium. eGFR